While much is known about hematopoietic stem (HSC) and progenitor cell (HPC) phenotype (cell surface and intracellular markers), heterogeneity, and function, there is still more to be learned, especially for how best to use and manipulate these cells for therapeutic advantage. The PI has run a continuously NIH-funded basic science laboratory for over 39 years. His goal continues to be to translate mechanistic insight for pre-clinical cell and animal models, and in collaboration with clinical investigators for clinical utility. The purpose of this NIH R35 application is to allow the PI and his lab ample uninterrupted time to pursue new ideas and creative approaches in context of two active NHLBI R01 grants. HL 056416 focuses on oxygen tension, metabolism, function and optimal collection and activities of HSCs from bone marrow (BM) and cord blood (CB), findings with implications for other stem and progenitor cell types. Exposure of mouse BM or human CB cells to ambient air within minutes induces Extra Physiological Shock Stress (EPHOSS), causing loss of HSCs through Reactive Oxygen Species (ROS) induced differentiation, not cell death. EPHOSS may have implications for personalized medicine to more accurately evaluate gene expression profiles and cell responsiveness in situations more closely related to O2 tension of cells in their in vivo microenvironment, compared to cells collected/processed in an artificial environment of ambient air. HL 112669 assesses effects of CD26/Dipeptidylpeptidase (DPP)4 in truncating selected cytokines (e.g. GM-CSF, G-CSF, IL-3, EPO, TPO) and chemokines (e.g. CXCL12, CCL3), thus changing their receptor-binding, cellular and intracellular activities. This has relevance to all systems in which cytokines/chemokines play a regulatory role. Blocking EPHOSS or inhibiting CD26/DPP4 enzymatic activity results in collection of increased numbers of HSCs. We will produce new information on mechanistic regulation of hematopoiesis. Our hypotheses and significance are: understanding EPHOSS and enzymatic activities of DPP4 and their manipulation will lead to a better understanding of HSC/HPC functions in health, ageing, and disease, information that can be used to accelerate patient recovery from stress/disease and for human CB hematopoietic cell transplantation (HCT). Our goals are to determine: 1) what hypoxic collection, processing, cell culture, and in vivo engraftment, and DPP4 inhibition, informs us about the regulation of HSCs, HPCs, and hematopoiesis during health, in response to stress, ageing and disease; 2) other means to compensate for effects of EPHOSS for more efficient HCT, and 3) differences in cell cycle status and gene and protein expression profile of HSCs and HPCs collected/processed in hypoxia/air and/or -/+ DPP4 inhibition. The innovation lies in paradigm changing concepts to be addressed with state-of-the-art methodologies. What we find can be translated to other cell sources for HCT, and for cells/procedures in the emerging field of regenerative medicine to enhance treatment efficacy.